Wolbachia are obligate intracellular bacteria and one of the great bacterial pandemics in the history of life. As germline specialists in millions of invertebrate species, Wolbachia manipulate sexual reproduction to increase the fitness of infected females, which are the transmitting sex of Wolbachia. The most common reproductive pathology, Cytoplasmic Incompatibility (CI), is expressed as a post-fertilization embryonic lethality in crosses between infected males and uninfected females. Wolbachia-infected females, however, rescue the paternally- delivered embryonic defects and thus gain a large fitness advantage over uninfected females. Despite several decades of intense research aimed at identifying Wolbachia's ability to encrypt the sperm genome and cause CI, the underlying genetic and molecular bases of CI remain elusive. Our lab is uniquely situated to solve this mystery as we recently identified, for the first time, two Wolbachia genes that induce CI-like lethality in embryos. These two genes, which we denote CI Candidate 1 (CIC1) and CI Candidate 2 (CIC2), are preferentially expressed in infected host testes, as expected for genes involved in CI. Moreover, transgene expression in Wolbachia-free host testes induces embryonic lethality. Most notably, the transgene-induced lethality is rescued by Wolbachia in eggs from infected females. Thus, CIC1 and CIC2 recapitulate the nature of CI. Here, we will validate these candidates as bona fide CI genes by benchmarking their modes of action against wild type CI induced by Wolbachia cells. In Aim 1, we will use fluorescent microscopy to compare the embryonic defects induced by Wolbachia transgenes with those induced by conventional Wolbachia infections. In Aim 2, we will test if the Wolbachia candidate genes are capable of rescuing CI induced either by the transgenes themselves or a native Wolbachia infection. In Aim 3, we will test whether CIC1 and CIC2 require Juvenile Hormone Inducible-26, a host protein that is partially required for CI. To the best of our knowledge, this project is the closest the field has come to validating candidate genes that cause CI. If successful, the scholarship will pioneer genetic studies of symbionts that induce reproductive pathogenesis, inform Wolbachia's efficacy and delivery as a tool to control diverse zoonotic diseases, and provide multiple lines of evidence for the discovery of the first CI genes.